Purification and characterization of a maltooligosaccharide-forming α-amylase from a new Bacillus subtilis KCC103

Abstract

A maltooligosaccharide-forming alpha-amylase was produced by a new soil isolate Bacillus subtilis KCC103. In contrast to other Bacillus species, the synthesis of alpha-amylase in KCC103 was not catabolite-repressed. The alpha-amylase was purified in one step using anion exchange chromatography after concentration of crude enzyme by acetone precipitation. The purified alpha-amylase had a molecular mass of 53 kDa. It was highly active over a broad pH range from 5 to 7 and stable in a wide pH range between 4 and 9. Though optimum temperature was 65-70 degrees C, it was rapidly deactivated at 70 degrees C with a half-life of 7 min and at 50 degrees C, the half-life was 94 min. The K (m) and V (max) for starch hydrolysis were 2.6 mg ml(-1) and 909 U mg(-1), respectively. Ca(2+) did not enhance the activity and stability of the enzyme; however, EDTA (50 mM) abolished 50% of the activity. Hg(2+), Ag(2+), and p-hydroxymercurybenzoate severely inhibited the activity indicating the role of sulfydryl group in catalysis. The alpha-amylase displayed endolytic activity and formed maltooligosaccharides on hydrolysis of soluble starch at pH 4 and 7. Small maltooligosaccharides (D2-D4) were formed more predominantly than larger maltooligosaccharides (D5-D7). This maltooligosaccharide forming endo-alpha-amylase is useful in bread making as an antistaling agent and it can be produced economically using low-cost sugarcane bagasse.